Extraction of genomic and plasmid DNA from mammalian cells

In this application of BioEcho’s EchoLUTION CellCulture DNA Kit, genomic DNA as well as plasmid DNA
has been extracted from cultured cells as part of a complex antibody display project. Up to 40 μg of
gDNA was extracted from 1x106 cultured human cells. Genomic DNA from increasing numbers of cells
was successfully amplified to demonstrate the functionality of nucleic acid fractions obtained from low
to high sample input amounts. DNA yields increased linearly between 15.000 and 2x106 cells. DNA
isolated from transfected cells was successfully transformed into E. coli showing that also plasmid DNA
can be isolated using the kit. The EchoLUTION Tissue DNA Micro Kit was used, too, and up to 11 μg of
genomic DNA were purified from fresh and stabilized human adenoid tissues.

Introduction

While DNA – and especially plasmid DNA – extraction
from bacterial cells is well investigated and many kit
solutions are available, DNA extraction from
mammalian cells remains a challenging and
comparably expensive procedure. For optimal quality
of the product DNA, the cells must meet certain
requirements in terms of number, preparation and
buffer ingredients.
The extraction of genomic and plasmid DNA can be
necessary for various different experiments and
assays. Applications like antibody epitope mapping,
cell surface display or characterization of stably
transfected cell lines often require the extraction of
DNA from mammalian cells.
In this application note, we describe the extraction of
whole genomic DNA from various cell lines cultivated
in cell culture and from human tissue samples. We also
extracted plasmid DNA that had been transiently
transfected into the cells. We assess DNA quality,
reproducibility and workload.

Experimental Protocol

Amplification of genomic DNA after extraction from
cultured or tissue cells

Cells of various mammalian cell lines were subjected
to DNA extraction using the BioEcho EchoLUTION
CellCulture DNA Kit. DNA was extracted from 1x106 cells
of human cell lines HEK293 (adherent), Expi-293
(suspension) and JeKo (suspension), as well as presorted,
transiently transfected HEK293 (HEK293+)
according to the kit protocol. Here, a titration between
15.000 and 2.000.000 cultured cells was run. In parallel,
DNA from 1x106 HEK293 and transfected HEK293 cells
(HEK293+) was extracted using a Silica kit.
Furthermore, genomic DNA was extracted from 10 μg
each of fresh or RNAlater-stabilized human adenoid
tissue using the EchoLUTION Tissue gDNA Micro Kit.

Extracted DNA was characterized with respect to purity
and yield using a NanoDrop spectrophotometer.
Corresponding volumes of the elution fractions from
HEK293 and HEK293+ extraction obtained with the
EchoLUTION and the Silica kits were visualized on a
0.8% agarose gel via GelRed staining.

Extraction of plasmid DNA from mammalian cultured
cells

Since total DNA is extracted from the cells using the kit,
plasmid DNA should also be found in the extracted
DNA fraction. Therefore, we transiently transfected
HEK293 cells with a vector carrying an Ampicillin
resistance cassette (AmpR). After DNA extraction from
1.000.000 cells, we transformed 10, 20, 40, and 80 ng of
extracted DNA, respectively, into E. coli XL1-Blue
chemically competent bacteria using heat shock at
42°C for 45 sec, incubated the bacteria for 30 min at
37°C, 650 rpm in SOC media and plated them onto
2xYT-GA Agar plates. After overnight incubation at 37°C,
colonies were counted.

Results

DNA concentrations, total yield and purity (A260 / A280)
are shown in Table 1.
DNA extracted from untransfected and transiently
transfected HEK293 cells with two preparation
technologies (EchoLUTION vs. Silica) was visualized in
an agarose gel (Figure 1). Intact high-molecular weight
DNA was obtained in both cases whereas the yield was
higher with the EchoLUTION kit.

Click onto the graphic to enlarge table!

Click onto the graphics to enlarge figure 1 and figure 2!

DNA was obtained in both cases whereas the yield was higher with the EchoLUTION kit.

Next, we wanted to know whether the extracted DNA
could be used for PCR. The AAVS-1 locus could be
amplified by 2-step nested PCR (30 cycles each) as
expected. Figure 2 shows the resulting DNA.
In order to study DNA quality when working with a low
amount of cells, we prepared a cell number series
ranging from 15.000 to 2.000.000 HEK293 cells. Table 2
shows DNA concentration, yield and purity.

DNA yield increased linearly over the titration range of
sample input (Fig. 3). Absorption ratios were below the
optimal value when less than 125.000 cells were
processed. A similar observation was made when DNA
was extracted with Silica methods (data not shown).

For the experiment shown in Table 2, a standard 30-
cycle PCR was conducted to see whether genomic loci
could still be amplified from DNA with a reduced A260 /
A280 ratio. 25 ng of template DNA were used in each
sample. Results are shown in Figure 4. The reduced
absorption ratio had no effect on PCR amplification.

After DNA extraction from 34.000 transiently
transfected HEK293 cells, resulting DNA was directly
transformed into chemically competent E. coli XL1-Blue
bacteria. After overnight
culturing, a total of 20 clones
could be recovered. Plasmid
content was correctly proven
by DNA sequencing.

Click onto the graphic to enlarge table!

Click onto the graphics to enlarge figure 3 and figure 4!

Discussion

Of the kits tested, the BioEcho
EchoLUTION series and its
novel isolation technology
offers the fastest and easiest
DNA extraction from a wide
range of mammalian cell
preparations and tissues. The
amount of extracted DNA
correlates with cell number when the same cell line is
used in all samples. We found that even low amounts
of DNA can be used for PCR-type applications. The
validity of A260 /A280 ratios at lower DNA concentrations
is somewhat questionable because of limitations and
variances of OD values of diluted DNA solutions (1).
This is confirmed by the higher concentrated DNA
preparation of the same experimental series where
DNA concentrations are at a range that can be safely
measured even though the sample mass load was
significantly higher than with the initial titration
points. In conclusion, OD measurements should
generally be complemented with functional studies in
order to accurately assess DNA quality (1). This is
underlined by the results shown in Figure 4 where PCR
performance was robust over the entire titration range
of amount of sample input. The absence of potentially
inhibitory reagents in the kit used probably
contributes to this robustness.

Additionally, the extraction of plasmid DNA from
mammalian cells is possible with the EchoLUTION
CellCulture DNA Kit without any additional preparation
steps. Even low amounts of DNA can be used for direct
transformation of extracted DNA into bacterial cells as
well as for amplification reactions. This might be
further enhanced by treatment with exonucleases or
other methods that increase the ratio of plasmid DNA
to genomic DNA, or by changing transformation
parameters (e. g., using electroporation).